Localization of Cell Receptor-Related Genes of SARS-CoV-2 in the Kidney through Single-Cell Transcriptome Analysis.

BACKGROUND: The new coronavirus (SARS-CoV-2), which has been responsible for the recent coronavirus disease 2019 (COVID-19) pandemic, uses the cell receptor angiotensin converting enzyme-2 (ACE2) for entry and the serine protease TMPRSS2 for spike (S) protein priming. Meanwhile, the presence of B0AT1 (SLC6A19) may prevent TMPRSS2 from accessing the cutting position on ACE2. Identifying the expression of these cell receptor-related genes of SARS-CoV-2 is critical for understanding the pathogenesis of SARS-CoV-2 in various tissues, especially in the kidney. METHODS: The single-cell transcription datasets of the human cell landscape (HCL) and other relevant single-cell transcription databases were used to analyze the expression of ACE2, TMPRSS2, and SLC6A19 in various organs and tissues, but mainly in the kidney. RESULTS: ACE2 was significantly expressed in the S1, S2, and S3 segments of proximal tubule (PT) cells. TMPRSS2 was widely expressed in several renal tubule populations extending from the PT cells to the collection system cell type, of which intercalated cells and the distal convoluted tubule cells showed more significant expression than PT cells. Unexpectedly, although expressed on various renal tubule populations, SLC6A19 was mainly enriched in PT cells, in line with ACE2 expression. Although alveolar-type (AT) 2 cells of the lung and intestinal epithelial cells expressed ACE2 as well as PT cells, AT 2 cells significantly expressed TMPRSS2 but not SLC6A19, while all 3 genes were significantly expressed in intestinal epithelial cells. CONCLUSIONS: ACE2 was widely expressed in specific cell subgroups of various human tissues, especially in intestinal epithelial cells, kidney PT cells, and also AT 2 cells of the lung. These 3 types of cells demonstrated significant differences in the distribution of the cell receptor-related genes of SARS-CoV-2, which may indicate the diversity of cell surface structures and differences in the affinity between SARS-CoV-2 and cells.

Allitridin inhibits human cytomegalovirus replication in vitro.

Human cytomegalovirus (HCMV) has been associated with a wide spectrum of diseases. There is currently no effective treatment for eliminating the virus. Garlic bulb extract has been reported to possess anti-viral efficacy. This study aimed to investigate the expression of the immediate­early (IE; ul122 and ul123), early (E; ul54) and late (L; ul83) genes of HCMV as well as the inhibitory effect of allitridin on the transcription levels of these genes. The results indicated that a HCMV gene expression cascade occurred, and that the deletion of IE72 had no influence on the transcription of the ul122 gene, while it led to significant reductions of ul54 and ul83 mRNA expression levels. Additionally, allitridin effectively suppressed the transcription of the HCMV IE, E and L genes; the inhibition rates of the transcription of the ul122 and ul123 genes were higher compared with those of ul54 and ul83 mRNA expression, while the expression of the IE genes was not significantly reduced by ganciclovir (GCV). Our results indicate that the HCMV IE72 deletion mutant strain affects the transcription of the virus downstream gene, allitridin inhibits HCMV infection in vitro, and that the IE genes may be the key target of allitridin in its action against HCMV.

Isolation and identification of chlorinated genistein from Actinoplanes sp. HBDN08 with antioxidant and antitumor activities.

A strain Actinoplanes sp. HBDN08 was screened by PCR-guided method using primers derived from conserved regions of halogenase genes. A new chlorinated isoflavone, 3',8-dichlorogenistein (1), along with 8-chlorogenistein (2) were isolated from the fermentation broth of Actinoplanes sp. HBDN08. Their structures were elucidated on the basis of extensive 1D and 2D NMR as well as HRESI-MS, ESI-MS, UV, and IR spectroscopic analyses. The origin of the two compounds was also investigated by high-performance liquid chromatography (HPLC) analysis. The results demonstrated that they were not biosynthesized but derived from the biotransformation of genistein by Actinoplanes sp. HBDN08. The antioxidant activities of the isolated compounds 1 and 2 were evaluated by using the lipid peroxidation assay. Their antitumor activities were calculated according to the inhibitory rate of cell proliferation against the human breast cancer cell line MDA-MB-231. The results indicated that compounds 1 (IC(50) = 5.2 microM) and 2 (IC(50) = 7.5 microM) showed stronger antioxidant activities than genistein (IC(50) = 13.6 microM). In comparison with the antitumor activities of genistein, those of compounds 1 and 2 increased 7.7- and 2.6-fold, respectively. These results suggest that the PCR-guided screening strategy is a rapid method for obtaining halometabolite-producing strains. Moreover, these results reveal that chlorination has significant effects on the bioactivities of genistein. This could be important information for studying the structure-activity relationships of genistein.

[Detection of human rhinovirus genes from clinical sample by one-step RT-PCR].

OBJECTIVE: Human rhinovirus (HRV) is the most common respiratory pathogen, which causes not only acute respiratory infection and community acquired pneumonitis in children, but also asthma episode and deterioration. However, the detection of respiratory pathogen, which mainly focuses on respiratory syncytial virus, influenzaviruses A and B, parainfluenza viruses 1-3 and adenoviruses, does not include HRV yet by now in China. The absence of detection method limits the clinical understanding of HRV pathogenicity, and causes unreasonable use of antibiotics. This study aimed to establish a one-step reverse transcription (RT) PCR system for detecting specific fragment of HRV RNA, and to analyze the sequences of amplicons. METHODS: A pair of degenerate primers based on the HRV highly conserved 5'' noncoding region (NCR) were used to develop a one-step RT-PCR system for detecting HRV RNA in nasopharyngeal aspirates from 78 children with acute respiratory tract infections in the spring of 2004. All the positive PCR products were sequenced, and the sequences of the nucleotides were analyzed by using biological software and compared with those in the GeneBank. RESULTS: Eleven (14.1%) of 78 samples were positive on RT-PCR, these patients were clinically diagnozed as upper respiratory tract infection (n = 7), bronchitis (n = 3) and bronchopneumonia (n = 1), respectively. Compared with the sequences of clinical and standard HRV viruses in the GeneBank, the nucleotide sequences of these 11 amplicons shared high homology of 89%-95.5%. Within the 11 amplicons, nucleotide identity varied from 75.2% to 91.8%, and the ratio of genetic variation was from 8.8% to 31.0%, which occurred in highly conserved regions and usually showed single nucleotide mutation in some special locations. These 11 amplicons attribute to the two branches of HRV cladogram, respectively. Most of mutations in highly conservative domain occurred on single ribonucleotide, mainly as transversion (C/G, A/G) and transition (T/C, A/G), some were mutations among 3 bases (A/C/G, A/T/G, A/C/T). And a few mutations involved two nearby ribonucleotide which were also found in highly conservative domain. However, ribonucleotide deletion and insertion were usually found in highly variable domain. CONCLUSION: The findings showed that this one-step RT-PCR system was highly specific, rapid and convenient for the detection of HRV RNA in nasopharyngeal secretions of patients with acute respiratory tract infections and that the genome of HRV viruses was highly variable.

[BALB/c mice model system of cytomegalovirus-induced myocarditis].

OBJECTIVE: To establish a BALB/c mice model system of cytomegalovirus-induced myocarditis. METHODS: Twenty five specific pathogen-free inbred female BALB/c mice (5 weeks old, 16 - 18 g, seronegative for MCMV) were infected with 1 x 10(4) PFU MCMV by the intraperitoneal (i.p.) route. All experimental mice were sacrificed at 3, 5, 7, 10, 14 days i.p. (n = 5 per time point). Hearts were removed under aseptic conditions, and were transected along the midline. One part of each heart was processed with Bouin's fixative for histological examination. The other part of each heart was immediately frozen in liquid nitrogen and stored at -80 degrees C until MCMV titre was determined by plaque assay. Serum cTnI level was assayed by ELISA. RESULTS: MCMV was detected in the hearts at extremely low levels on 3 days i.p. and could not be detected on 10 days i.p. A mixed cellular infiltrate composed of polymorphonuclear neutrophils and mononuclear lymphocytes was observed on 3 days, which reached a peak at 7 to 10 days after MCMV infection and was maintained for at least 3 - 4 months postinfection. Serum cTnI levels were elevated on 3 days i.p., reaching a peak at 7 to 10 days i.p.. CONCLUSIONS: These data highlight the possible therapeutic uses of antiviral drugs in viral myocarditis as well as further elucidating the pathogenic nature of the disease.